ABSTRACT
OBJECTIVE: RNAe is a new method that enhances protein expression at the post-transcriptional level. RNAe utility was further explored to improve endogenous protein expression. RESULTS: Transgenic mice were created by targeting RNAe to growth hormone gene into the C57/BL mouse genome by transposon mediated integration; the mice showed a heavier body weight and longer body length compared with normal mice. RNAe can also be used for gene therapy through the delivery of in vitro transcribed RNA. CONCLUSION: This study takes a further step towards applying RNAe in pharmaceutical approaches by transposon-based transgenic mice model construction and the use of in vitro transcribed RNA transfection assay.
Subject(s)
Growth Hormone/genetics , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , Animals , Cell Line , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Genetic Therapy/methods , HeLa Cells , Humans , Male , Mice , Mice, Transgenic , PregnancyABSTRACT
The growth plate (GP) comprising sequentially differentiated cell layers is a critical structure for bone elongation and regeneration. Although several key regulators in GP development have been identified using genetic perturbation, systematic understanding is still limited. Here, we used single-cell RNA-sequencing (RNA-seq) to determine the gene expression profiles of 217 single cells from GPs and developed a bioinformatics pipeline named Sinova to de novo reconstruct physiological GP development in both temporal and spatial high resolution. Our unsupervised model not only confirmed prior knowledge, but also enabled the systematic discovery of genes, potential signal pathways, and surface markers CD9/CD200 to precisely depict development. Sinova further identified the effective combination of transcriptional factors (TFs) that regulates GP maturation, and the result was validated using an in vitro EGFP-Col10a screening system. Our case systematically reconstructed molecular cascades in GP development through single-cell profiling, and the bioinformatics pipeline is applicable to other developmental processes. VIDEO ABSTRACT.
Subject(s)
Growth Plate/embryology , Growth Plate/metabolism , Sequence Analysis, RNA/methods , Signal Transduction/genetics , Single-Cell Analysis/methods , Animals , Animals, Newborn , Biomarkers/metabolism , Body Patterning/genetics , Flow Cytometry , Gene Regulatory Networks , Mice, Inbred C57BL , Reproducibility of Results , Time Factors , Transcription Factors/metabolismABSTRACT
In this study, a universal protein expression enhancement RNA tool, termed RNAe, was developed by modifying a recently discovered natural long non-coding RNA. At the moment, RNAe is the only technology for gene expression enhancement, as opposed to silencing, at the post-transcriptional level. With this technology, an expression enhancement of 50-1000% is achievable, with more than 200% enhancement achieved in most cases. This work identified the sufficient and necessary element for RNAe function, which was found to be merely 300 nucleotides long and was named minRNAe. It contains a 72-nt 5' pairing sequence which determines the specificity, a 167-nt short non-pairing interspersed nuclear element (SINE) B2 sequence which enhances ribosome recruitment to the target mRNA, and a poly(A) tail, provided together on a plasmid bearing the appropriate sequences. Cellular delivery of RNAe was achieved using routine transfection. The RNAe platform was validated in several widely-used mammalian cell lines. It was proven to be efficient and flexible in specifically enhancing the expression of various endogenous and exogenous proteins of diverse functions in a dose-dependent manner. Compared to the expression-inhibitory tool RNAi, the RNAe tool has a comparable effect size, with an enhancing as opposed to inhibitory effect. One may predict that this brand new technology for enhancing the production of proteins will find wide applications in both research and biopharmaceutical production.
Subject(s)
Gene Expression Regulation , Protein Biosynthesis , Protein Engineering/methods , RNA, Long Noncoding/chemistry , Antibody Formation , Cell Line , Genetic Vectors , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , HEK293 Cells , Humans , Proteomics , RNA, Antisense/chemistry , Repetitive Sequences, Nucleic Acid , Ribosomes/metabolismABSTRACT
Mesenchymal stem cells (MSCs) show great promise in blood vessel restoration and vascularization enhancement in many therapeutic situations. Typically, the co-implantation of MSCs with vascular endothelial cells (ECs) is effective for the induction of functional vascularization in vivo, indicating its potential applications in regenerative medicine. The effects of MSCs-ECs-induced vascularization can be modeled in vitro, providing simplified models for understanding their underlying communication. In this article, a contact coculture model in vitro and an RNA-seq approach were employed to reveal the active crosstalk between MSCs and ECs within a short time period at both morphological and transcriptional levels. The RNA-seq results suggested that angiogenic genes were significantly induced upon coculture, and this prevascularization commitment might require the NF-κB signaling. NF-κB blocking and interleukin (IL) neutralization experiments demonstrated that MSCs potentially secreted IL factors including IL1ß and IL6 to modulate NF-κB signaling and downstream chemokines during coculture. Conversely, RNA-seq results indicated that the MSCs were regulated by the coculture environment to a smooth muscle commitment within this short period, which largely induced myocardin, the myogenic co-transcriptional factor. These findings demonstrate the mutual molecular mechanism of MSCs-ECs-induced prevascularization commitment in a quick response.
